Microbial rhodopsins, a diverse group of photoactive proteins found in Archaea, Bacteria, and Eukarya, function in photosensing and photoenergy harvesting and may have been present in the resource-limited early global environment. Four different physiological functions have been identified and characterized for nearly 5,000 retinal-binding photoreceptors, these being ion transporters that transport proton or chloride and sensory rhodopsins that mediate light-attractant and/or -repellent responses. The greatest number of rhodopsins previously observed in a single archaeon had been four. Here, we report a newly discovered sixrhodopsin system in a single archaeon, Haloarcula marismortui, which shows a more diverse absorbance spectral distribution than any previously known rhodopsin system, and, for the first time, two light-driven proton transporters that respond to the same wavelength. All six rhodopsins, the greatest number ever identified in a single archaeon, were first shown to be expressed in H. marismortui, and these were then overexpressed in Escherichia coli. The proteins were purified for absorption spectra and photocycle determination, followed by measurement of ion transportation and phototaxis. The results clearly indicate the existence of a proton transporter system with two isochromatic rhodopsins and a new type of sensory rhodopsin-like transducer in H. marismortui.Microbial rhodopsins comprise a large family of seven-transmembrane helical proteins that either mediate light-driven ion transport to harvest solar energy or serve as receptors to mediate phototaxis (13) and possibly photoadaptation (32). In archaea, four rhodopsins responding to different wavelengths of light with distinct functions in Halobacterium salinarum have been identified and characterized (20,32 The two ion-transporting rhodopsins perform light-driven outward proton transport to create a proton-electrochemical potential or inward chloride transport to maintain the osmotic and pH homeostasis of the cell. The photoactivated sensory rhodopsins, on the other hand, undergo light-triggered conformational changes to relay signals to their cognate transducers and consequently activate signaling cascades in a manner similar to that of the two-component system involved in eubacterial chemotaxis (1, 22) to control flagellum rotation and thus swimming direction. Our current understanding of microbial rhodopsins as both ion transporters and photosensory receptors has been based primarily on these four known rhodopsins.A recently completed genome project for Haloarcula marismortui (3) proposed the existence of six opsin-related genes, the greatest number ever found in a single archaeon. This proposal immediately raised three questions. (i) Are these six rhodopsins biologically expressed and functionally active? (ii) What is the maximum-absorbance-wavelength ( max ) distribution pattern of these six rhodopsins? (iii) Do the two extra rhodopsins, compared to the four in H. salinarum, perform new functions or have new features beyond those of t...
The haem‐containing mono‐oxygenase cytochrome P450 3A4 (CYP3A4) and its redox partner NADPH‐dependent cytochrome P450 oxidoreductase (CPR) are among the most important enzymes in human liver for metabolizing drugs and xenobiotic compounds. They are membrane‐bound in the endoplasmic reticulum (ER). How ER colocalization and the complex ER phospholipid composition influence enzyme activity are not well understood. CPR and CYP3A4 were incorporated into phospholipid bilayer nanodiscs, both singly, and together in a 1 : 1 ratio, to investigate the significance of membrane insertion and the influence of varying membrane composition on steady‐state reaction kinetics. Reaction kinetics were analysed using a fluorimetric assay with 7‐benzyloxyquinoline as substrate for CYP3A4. Full activity of the mono‐oxygenase system, with electron transfer from NADPH via CPR, could only be reconstituted when CPR and CYP3A4 were colocalized within the same nanodiscs. No activity was observed when CPR and CYP3A4 were each incorporated separately into nanodiscs then mixed together, or when soluble forms of CPR were mixed with preassembled CYP3A4‐nanodiscs. Membrane integration and colocalization are therefore essential for electron transfer. Liver microsomal lipid had an enhancing effect compared with phosphatidylcholine on the activity of CPR alone in nanodiscs, and a greater enhancing effect on the activity of CPR‐CYP3A4 nanodisc complexes, which was not matched by a phospholipid mixture designed to mimic the ER composition. Furthermore, liver lipid enhanced redox coupling within the system. Thus, natural ER lipids possess properties or include components important for enhanced catalysis by CPR‐CYP3A4 nanodisc complexes. Our findings demonstrate the importance of using natural lipid preparations for the detailed analysis of membrane protein activity.
Caveolae are small cell surface invaginations, important for control of membrane tension, signaling cascades and lipid sorting. Their formation is coupled to the lipid-dependent oligomerization of the proteins Caveolin1 and Cavin1, which are essential for membrane curvature generation. Yet, the mechanistic understanding of how Cavin1 assembles at the membrane interface is lacking. Here, we used model membranes combined with biophysical techniques to show that Cavin1 inserts into membranes. We found that the helical region 1 (HR1) initiated membrane binding via electrostatic interactions, which is further enforced by partial helical insertion in a PI(4,5)P2-dependent process mediated by the disordered region 1 (DR1). In agreement with this, the DR1 was found important for the co-assembly of Cavin1 with Caveolin1 in living cells. We propose that DR1 and HR1 of Cavin1 constitute a novel membrane interacting unit facilitating dynamic rounds of assembly and disassembly of Cavin1 at the membrane.
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